Efforts to understand the complex relationship between microbes and their hosts are complicated by the large number of nonculturable organisms, and the heterogeneity even within each species. While modern metagenomics approaches admirably sample the identity of microbes, bulk studies limit the other inferences that can be derived from each bacteria. In order to overcome this problem, yet retain clues to the diversity of the original population, we propose to: Model, design, build, and test a multidimensional, microfluidic sorter based on both structural (size-and shape and Electrophoresis) and functional (Adhesion, Chemotaxis) parameters to separate a complex bacterial mixture into bins containing bacteria that share common properties. The identities of the sorted bacteria will be obtained through metagenomic studies. The device will enable determination of the heterogeneity both between and within species in a complex mixture. This microfluidic separation device will utilize 1. Asymmetric pinched flow fractionation to separate bacteria based on size and shape. 2. Electrophoretic based flow fractionation to separate bacteria based on surface charge. 3. Functionalized magnetic beads to separate bacteria based on adhesion to extracellular matrix (ECM) components. 4. Chemotaxis to separate bacteria based on their motile response to chemical stimuli, and lastly, 5. Multi separation modalities to separate bacteria based on size, shape, adhesion, response to chemical stimuli, and surface charge. We will use microfluidic approaches since (i) the feature sizes of microfluidic systems are compatible with the size of the bacteria;(ii) complicated flow paths can be machined with ease and at low cost;(iii) many sorting modules utilizing diverse principles can be integrated into a single device. Within each specific aim we rely heavily on direct numerical simulation of particle movement using code that reflects 2-dimensional geometry. As part of the experimental plan, we will expand functionality of the our custom Particle Mover program to a full 3-D simulation. Once design parameters have been established, devices will be fabricated and tested rigorously using particles, mixtures of known bacteria, and for the 3 and 4-stage devices, complex mixtures from human subjects. We will make use of a modular architecture that facilitates interchangeability of modules. The long term goal is to add other separation modalities into the device and to integrate into the device modules for single cell isolation, DNA isolation and amplification on-chip, to permit high-throughput analysis of complex mixtures. These studies will lead to devices that not only capture the diversity of complex mixtures, but also permit direct assignment of the heterogeneity of structural and functional properties, genes and gene products within each single species in the mixture, and aid understanding of human disease. PUBLIC HEALTH RELEVANCE: This proposal represents a new collaborative effort between three established scientists with unique and complementary interests. By focusing our expertise in fluid dynamics (Hu), microfluidic design (Bau), and clinical and experimental bacterial infection (Worthen) we propose to: Model, design, build, and test a multidimensional, microfluidic sorter based on both structural (size-and shape and Electrophoresis) and functional (Adhesion, Chemotaxis) parameters to separate a complex bacterial mixture into bins containing bacteria that share common properties. The identities of the sorted bacteria will be obtained through metagenomic studies. The device will enable determination of the heterogeneity both between and within species in a complex mixture, such as in clinical infectious illnesses (we are particularly interested in Bronchiectasis and necrpotizing enterocolitis) whose pathogenesis is obscure. We also are interested in contributing to an understanding of how tools such as fluid dynamics (for which a new program of numerical simulation will be presented to the scientific community) and microfluidics intersect with medicine.